PCT/US90/01515 describes products and processes for gene therapy and gene immunization by direct introduction of naked or lipid-complexed polynucleotides into body tissue of a host vertebrate. (All references cited hereunder are incorporated herein by reference.) The naked polynucleotides are naked in the sense that they are free of certain indicated additives, such as transfection-facilitating proteins, viral particles, liposomes, charged lipids, and calcium phosphate precipitating agents. The lipid-complexed polynucleotides are formed of polynucleotides and lipids.
PCT/US94/06734 describes the work by Gossen, M. & Bujard, H. Proc Natl Acad Sci USA 89, 5547-5551 (1992). See also Gossen, M., Bonin, A. L. & Bujard, H. TIBS 18, 471-475 (1993). (All references cited hereunder are incorporated herein by reference.) Gossen and Bujard adapted a prokaryotic tetracycline system to produce a genetic switch for achieving control of eukaryotic gene expression. In the native prokaryotic tetracycline system, tetracycline is an effector that induces prokaryotic gene expression. Tetracycline accomplishes this by binding to a tetracycline repressor protein. In the absence of tetracycline, the tetracycline repressor binds a tetracycline operator sequence, which is linked to a promoter, and represses transcription. In the presence of tetracycline, the tetracycline repressor binds tetracycline, which binding displaces the repressor from the tetracycline operator sequence, so repression is relieved and transcription can begin.
The Gossen and Bujard adaptation, instead of being a tetracycline repressor system, is a tetracycline activator system. In this system, a tetracycline-controlled activator protein is prepared by fusing the tetracycline repressor to a transcription activation domain from another protein that activates transcription in eukaryotic cells, causing the resultant chimeric protein to retain the repressor's binding capabilities, while also possessing the property of activating transcription in eukaryotic cells. In the absence of tetracycline, the tetracycline-controlled activator binds the tetracycline operator sequence, which is linked to a promoter, and activates transcription. In the presence of tetracycline, the tetracycline-controlled activator binds tetracycline, which binding displaces the activator from the tetracycline operator sequence, so activation is ended and transcription is silenced.
According to Furth, P. A., et al. Proc Natl Acad Sci USA 91, 9302-9306 (1994), the Gossen and Bujard group generated a transgenic mouse based on the tetracycline activator system. In Gatz, C., Frohberg, C. & Wendenburg, R. Plant J 2, 397-404 (1992), this investigative group produced a transgenic plant using the tetracycline repressor system. Each regulatory system exploits the prokaryotic tetracycline mechanism as a genetic switch to turn genes on and off.
These regulatory systems require that two different kinds of expression vectors enter each transfected cell. For example, in the tetracycline activator system, a first expression vector is necessary that encodes the tetracycline-controlled activator. A second expression vector is required that encodes the gene of interest placed under the control of the tetracycline operator sequence linked to a promoter.
Similarly, in the tetracycline repressor system, the tetracycline repressor is encoded by one expression vector. The gene of interest is encoded by another expression vector placed under the control of the tetracycline operator sequence linked to a promoter.
Such two-vector systems are, however, problematic. The probability of transfecting a single cell with two plasmid DNAs is significantly lower than for transfecting that cell with one plasmid DNA. Moreover, the transfection efficiencies for different kinds of plasmid DNAs may vary dramatically. Furthermore, even where multiple copies of plasmid DNA get transfected into cells, the sheer number of plasmid DNAs is presumed to affect the level of expression, making it important for regulation of that expression to have both kinds of plasmid DNAs available in sufficiently representative quantities.
PCT/US94/06734 tried to solve the two-vector problem by eliminating the need to encode one of the genes, to wit, the gene of interest. A DNA construct was designed for homologous recombination at a single site in a haploid genome. This construct encoded only the tetracycline-controlled activator and placed the tetracycline operator sequence linked to a promoter in a position so that, upon successful integration, it controlled the expression of an endogenous gene. Successful integration was also intended to position the sequence encoding the tetracycline-controlled activator under the control of the promoter of that endogenous gene.
This single construct system served the purpose of generating a conditional gene knockout. Functioning as a single copy, in an integrated state, at a predetermined locus, this plasmid was not developed to shuttle individual genes into cells, in contrast to gene therapy and gene immunization described in PCT/US90/01515 where plasmids are present as multiples copies, in episomal form, and for the express purpose of gene delivery. Additionally, this single plasmid solution was limited to the production of organisms as "transgenics," given the technical manipulations required to bring about homologous recombination, thus effectively precluding its usefulness in gene therapy and gene immunization in humans.
Even this single construct solution fails to overcome additional existing difficulties. It is critical that expression not be leaky in these regulatory systems that seek to control gene activity. To ensure this, PCT/US94/06734 contemplated insulating one transcription unit, constituting the endogenous gene and the tetracycline response element, from the other transcription unit, having the sequence encoding the tetracycline-controlled activator under the control of the endogenous promoter. For example, strong transcription terminators were envisioned for placement between these transcription units.
Now that individual genes can be administered as drugs and vaccines for use in gene therapy and gene immunization, there arises an urgent and compelling need to provide delivery of these genes and regulate expression of their gene products. The realization of this goal through the development of regulatory systems should take into account the problems of two-plasmid systems. It would be particularly beneficial if any such regulatory systems could address the issue of leaky gene expression toward achieving genetic control.
Accordingly, it is an objective of the invention to provide effector controlled eukaryotic expression vectors adapted for use in gene therapy and gene immunization.
It is another objective of the invention to provide these vectors as single vector constructs that contain all the elements necessary for a regulatory system on a single construct.
It is yet another objective of the invention to provide such vectors as having positive feedback regulation.
These and other objectives of the invention will be apparent upon consideration of the specification as a whole.